Early hepatic microvascular injury in response to acetaminophen toxicity

OBJECTIVE: The hepatic toxic response to acetaminophen (APAP) is characterized by centrilobular (CL) necrosis preceded by hepatic microvascular injury and congestion. The present study was conducted to examine changes in liver microcirculation after APAP dosing. METHODS: Male C57Bl/6 mice were treated with APAP (600 mg/kg body weight) by oral gavage. The livers of anesthetized mice were examined using established in vivo microscopic methods at 0, 0.5, 1, 2, 4, 6, 12 hours after APAP. RESULTS: The levels of hepatic transaminases (i.e., alanine aminotransferase [ALT] and aspartate transaminase) increased minimally for up to 2 hours. Thereafter, their levels were significantly and progressively increased. The numbers of swollen sinusoidal endothelial cells (SECs) in periportal regions were increased (3.5-fold) from 0.5 to 6 hours, and those in CL regions were increased (4.0-fold) at 0.5 and 1 hour. The intensity of in vivo staining for formaldehyde-treated serum albumin, which is a specific ligand for SECs, was reduced from 2 to 12 hours. Erythrocytes infiltrated into the space of Disse as early as 2 hours, and the area occupied by these cells was markedly increased at 6 hours. Sinusoidal perfusion was reduced from 1 through 12 hours, with a nadir (35% decrease) at 4 and 6 hours. Phagocytic Kupffer cell activity was significantly elevated from 0.5 through 12 hours. Although gadolinium chloride minimized the changes in sinusoidal blood flow and reduced ALT levels 6 hours after APAP, it failed to inhibit endothelial swelling, extravasation of erythrocytes, and CL parenchymal necrosis. CONCLUSIONS: These results confirm that APAP-induced SEC injury precedes hepatocellular injury, supporting the hypothesis that SECs are an early and direct target for APAP toxicity. These findings also suggest that reduced sinusoidal perfusion and increased Kupffer cell activity contribute to the development of APAP-induced liver injury.     

Neutrophil depletion protects against murine acetaminophen hepatotoxicity

We previously reported that liver natural killer (NK) and NKT cells play a critical role in mouse model of acetaminophen (APAP)-induced liver injury by producing interferon gamma (IFN-gamma) and modulating chemokine production and subsequent recruitment of neutrophils into the liver. In this report, we examined the role of neutrophils in the progression of APAP hepatotoxicity. C57BL/6 mice were given an intraperitoneal toxic dose of APAP (500 mg/kg), which caused severe acute liver injury characterized by significant elevation of serum ALT, centrilobular hepatic necrosis, and increased hepatic inflammatory cell accumulation. Flow cytometric analysis of isolated hepatic leukocytes demonstrated that the major fraction of increased hepatic leukocytes at 6 and 24 hours after APAP was neutrophils (Mac-1+ Gr-1+). Depletion of neutrophils by in vivo treatment with anti-Gr-1 antibody (RB6-8C5) significantly protected mice against APAP-induced liver injury, as evidenced by markedly reduced serum ALT levels, centrilobular hepatic necrosis, and improved mouse survival. The protection was associated with decreased FasL-expressing cells, cytotoxicity against hepatocytes, and respiratory burst in hepatic leukocytes. In intracellular adhesion molecule (ICAM)-1-deficient mice, APAP caused markedly reduced liver injury when compared with wild-type mice. The marked protection in ICAM-1-deficient mice was associated with decreased accumulation of neutrophils in the liver. Hepatic GSH depletion and APAP-adducts showed no differences among the antibody-treated, ICAM-1-deficient, and normal mice. In conclusion, accumulated neutrophils in the liver contribute to the progression and severity of APAP-induced liver injury.     

Role of hepatic fibrin in idiosyncrasy-like liver injury from lipopolysaccharide-ranitidine coexposure in rats

Coadministration of nonhepatotoxic doses of the histamine 2-receptor antagonist ranitidine (RAN) and bacterial lipopolysaccharide (LPS) results in hepatocellular injury in rats, the onset of which occurs in 3 to 6 hours. This reaction resembles RAN idiosyncratic hepatotoxicity in humans. Early fibrin deposition occurs in livers of rats cotreated with LPS/RAN. Accordingly, we tested the hypothesis that the hemostatic system contributes to liver injury in LPS/RAN-treated rats. Rats were given either LPS (44.4 x 10(6) EU/kg) or its vehicle, then RAN (30 mg/kg) or its vehicle 2 hours later. They were killed 2, 3, 6, 12, or 24 hours after RAN treatment, and liver injury was estimated from serum alanine aminotransferase activity. A modest elevation in serum hyaluronic acid, which was most pronounced in LPS/RAN-cotreated rats, suggested altered sinusoidal endothelial cell function. A decrease in plasma fibrinogen and increases in thrombin-antithrombin dimers and in serum concentration of plasminogen activator inhibitor-1 occurred before the onset of liver injury. Hepatic fibrin deposition was observed in livers from LPS/RAN-cotreated rats 3 and 6 hours after RAN. Liver injury was abolished by the anticoagulant heparin and was significantly attenuated by the fibrinolytic agent streptokinase. Hypoxia, one potential consequence of sinusoidal fibrin deposition, was observed in livers of LPS/RAN-treated rats. In conclusion, the results suggest that the hemostatic system is activated after LPS/RAN cotreatment and that fibrin deposition in liver is important for the genesis of hepatic parenchymal cell injury in this model.     

Ethanol binging enhances hepatic microvascular responses to acetaminophen in mice

OBJECTIVE: Chronic alcoholism has been considered to be a risk for acetaminophen (APAP) hepatotoxicity, but little is known about the effect of binge alcohol drinking on APAP-induced liver injury. The present study was conducted to examine the effect of ethanol binging on APAP-induced hepatic microcirculatory dysfunction. METHODS: Male C57Bl/6 mice received 3 weekly ethanol binges (4 g/kg every 12 h x 5 doses/ week) or water binges. At 12 h after the last gavage, APAP (300 mg/kg) was given by oral gavage. In one group of mice, gadolinium chloride (GdCl3, 10 mg/kg) was intraperitoneally administered 2 and 1 days before the start of each weekly ethanol binge. RESULTS: Ethanol binging enhanced APAP-induced liver injury as indicated by ALT levels. Intravital microscopic study showed that APAP further increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space as well as Kupffer cell phagocytic activity in ethanol-binged mice when compared with water-binged mice, while no significant differences in sinusoidal perfusion and leukocyte adhesion were observed. ALT levels after APAP were exacerbated in ethanol-binged mice treated with GdCl3, but APAP-induced hepatic microcirculatory dysfunction was not changed significantly. CONCLUSIONS: These results suggest that ethanol binging increases APAP-induced liver injury by exacerbating infiltration of the Disse space with blood cells. Kupffer cells exert a protective role in the liver against APAP intoxication following ethanol binging.     

Mild hypothermia attenuates liver injury and improves survival in mice with acetaminophen toxicity

BACKGROUND & AIMS: Body temperature may critically affect mechanisms of liver injury in acetaminophen (APAP) hepatotoxicity. In addition, mild hypothermia is used to treat intracranial hypertension in human liver failure without detailed information on its effects on the injured liver itself. Therefore, we investigated the effects of body temperature on the progression of APAP-induced liver injury in mice. METHODS: Male C57BL6 mice treated with saline or APAP (300 mg/kg intraperitoneally) were maintained at normothermia (35.5-37.5 degrees C) by external warming or were allowed to develop mild hypothermia (32.0-35.0 degrees C) after 2 hours from APAP administration. RESULTS: Mild hypothermia resulted in improved survival after APAP intoxication. Liver damage was reduced, as assessed histologically and by plasma alanine aminotransferase levels. Early effects of hypothermia included a reduction of hepatic congestion and improved recovery of glycogen stores. At later time points (8-12 hours), APAP-treated mice that were maintained at normothermia manifested increased hepatocyte apoptosis, as assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining and cleavage of poly(adenosine diphosphate-ribose) polymerase. Mild hypothermia did not affect the formation of APAP-protein adducts or the depletion of glutathione, nor did it abrogate hepatocyte DNA synthesis. CONCLUSIONS: Mild hypothermia improved survival and attenuated liver injury and apoptosis in APAP-treated mice by reducing hepatic congestion and improving glycogen recovery without affecting hepatic regeneration. Results of the study underscore the need for a strict control of body temperature in animal models of liver failure and suggest that the benefits of mild hypothermia in liver failure may extend beyond those related to reduced cerebral complications.     

Mitochondrial protection by the JNK inhibitor leflunomide rescues mice from acetaminophen-induced liver injury

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses but which can precipitate liver injury at high doses. We have previously found that the antirheumatic drug leflunomide is a potent inhibitor of APAP toxicity in cultured human hepatocytes, protecting them from mitochondria-mediated cell death by inhibiting the mitochondrial permeability transition. The purpose of this study was to explore whether leflunomide protects against APAP hepatotoxicity in vivo and to define the molecular pathways of cytoprotection. Male C57BL/6 mice were treated with a hepatotoxic dose of APAP (750 mg/kg, ip) followed by a single injection of leflunomide (30 mg/kg, ip). Leflunomide (4 hours after APAP dose) afforded significant protection from liver necrosis as assessed by serum ALT activity and histopathology after 8 and 24 hours. The mechanism of protection by leflunomide was not through inhibition of cytochrome P450 (CYP)-catalyzed APAP bioactivation or an apparent suppression of the innate immune system. Instead, leflunomide inhibited APAP-induced activation (phosphorylation) of c-jun NH2-terminal protein kinase (JNK), thus preventing downstream Bcl-2 and Bcl-XL inactivation and protecting from mitochondrial permeabilization and cytochrome c release. Furthermore, leflunomide inhibited the APAP-mediated increased expression of inducible nitric oxide synthase and prevented the formation of peroxynitrite, as judged from the absence of hepatic nitrotyrosine adducts. Even when given 8 hours after APAP dose, leflunomide still protected from massive liver necrosis. Conclusion: Leflunomide afforded protection against APAP-induced hepatotoxicity in mice through inhibition of JNK-mediated activation of mitochondrial permeabilization.     

Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity

BACKGROUND & AIMS: Inflammatory mediators released by nonparenchymal inflammatory cells in the liver have been implicated in the progression of acetaminophen (APAP) hepatotoxicity. Among hepatic nonparenchymal inflammatory cells, we examined the role of the abundant natural killer (NK) cells and NK cells with T-cell receptors (NKT cells) in APAP-induced liver injury. METHODS: C57BL/6 mice were administered a toxic dose of APAP intraperitoneally to cause liver injury with or without depletion of NK and NKT cells by anti-NK1.1 monoclonal antibody (MAb). Serum alanine transaminase (ALT) levels, liver histology, hepatic leukocyte accumulation, and cytokine/chemokine expression were assessed. RESULTS: Compared with APAP-treated control mice, depletion of both NK and NKT cells by anti-NK1.1 significantly protected mice from APAP-induced liver injury, as evidenced by decreased serum ALT level, improved survival of mice, decreased hepatic necrosis, inhibition of messenger RNA (mRNA) expression for interferon-gamma (IFN-gamma), Fas ligand (FasL), and chemokines including KC (Keratinocyte-derived chemokine); MIP-1 alpha (macrophage inflammatory protein-1 alpha); MCP-1 (monocyte chemoattractant protein-1); IP-10 (interferon-inducible protein); Mig (monokine induced by IFN-gamma) and decreased neutrophil accumulation in the liver. Hepatic NK and NKT cells were identified as the major source of IFN-gamma by intracellular cytokine staining. APAP induced much less liver injury in Fas-deficient (lpr) and FasL-deficient (gld) mice compared with that in wild-type mice. CONCLUSIONS: NK and NKT cells play a critical role in the progression of APAP-induced liver injury by secreting IFN-gamma, modulating chemokine production and accumulation of neutrophils, and up-regulating FasL expression in the liver, all of which may promote the inflammatory response of liver innate immune system, thus contributing to the severity and progression of liver injury downstream of the metabolism of APAP and depletion of reduced glutathione (GSH) in hepatocytes.     

Dietary steatotic liver attenuates acetaminophen hepatotoxicity in mice

OBJECTIVE: To determine whether hepatic steatosis is susceptible to acetaminophen (APAP) hepatotoxicity. METHODS: Male C57Bl/6 mice were fed a "Western-style" diet (high fat and high carbohydrate) for 4 months to develop severe hepatic steatosis with mild increases in alanine aminotransferase (ALT) levels. These were compared to mice fed a standard chow diet. RESULTS: Treatment with APAP (300 mg/kg, orally) to mice fed a regular chow increased ALT levels (519-fold) and caused hepatic centrilobular injury at 6 h. APAP increased hepatic cytochrome-P (CYP)-2E1 mRNA levels (17-fold). In vivo microscopic studies showed that APAP caused a 30% decrease in sinusoidal perfusion and the infiltration of red blood cells into the space of Disse. Electron microscopy demonstrated that numerous gaps were formed in sinusoidal endothelial cells. Mice fed the "Western-style" diet were protected from APAP hepatotoxicity as evidenced by 89% decrease in ALT levels and less centrilobular injury, which was associated with 42% decrease in CYP2E1 mRNA levels. The APAP-induced liver microcirculatory dysfunction was minimized in mice fed the "Western-style" diet. CONCLUSIONS: These results suggest that hepatic steatosis elicited by the "Western-style" diet attenuated APAP-induced hepatotoxicity by inhibiting CYP2E1 induction and by minimizing sinusoidal endothelial cell injury, leading to protection of liver microcirculation.     

Mechanisms of protection by melatonin against acetaminophen-induced liver injury in mice

The present study was performed to determine whether melatonin protects mouse liver against severe damage induced by acetaminophen (APAP) administration and where melatonin primarily functions in the metabolic pathway of APAP to protect mouse liver against APAP-induced injury. Treatment of mice with melatonin (50 or 100 mg/kg, p.o.) 8 or 4 hr before APAP administration (750 mg/kg, p.o.) suppressed the increase in plasma alanine aminotransferase and aspartate aminotransferase activities in a dose- and a time-dependent manner. Melatonin treatment (100 mg/kg, p.o.) 4 hr before APAP administration remarkably inhibited centrilobular hepatic necrosis with inflammatory cell infiltration and increases in hepatic lipid peroxidation and myeloperoxidase activity, an index of tissue neutrophil infiltration, as well as release of nitric oxide and interleukin-6 into blood circulation at 9 hr after APAP administration. However, melatonin neither affected hepatic reduced glutathione (GSH) content nor spared hepatic GSH consumption by APAP treatment. Moreover, pretreatment with melatonin 4 hr before APAP administration did not influence the induction of hepatic heat shock protein 70 (HSP70) by APAP and melatonin alone did not induce HSP70 in mouse liver. These results indicate that exogenously administered melatonin exhibits a potent hepatoprotective effect against APAP-induced hepatic damage probably downstream of the activity of cytochrome P450 2E1, which works upstream of GSH conjugation in the pathway of APAP metabolism, via its anti-nitrosative and anti-inflammatory activities in addition to its antioxidant activity.     

Tissue factor deficiency and PAR-1 deficiency are protective against renal ischemia reperfusion injury

Ischemia/reperfusion (IR) injury is a leading cause of acute renal failure and an important contributor to allograft damage. Tissue factor (TF) is up-regulated during IR, and TF inhibition reduces renal injury. However, the underlying mechanisms by which TF contributes to injury have not been elucidated. We postulated that TF contributes to IR injury by production of coagulation proteases and subsequent signaling by protease activated receptor (PARs). We compared renal injury after 25 minutes of bilateral renal ischemia and varying periods of reperfusion in C57BL/6 mice, those expressing low levels of TF (low-TF), hirudin-treated C57BL/6, and mice lacking either PAR-1 or PAR-2. C57BL/6 mice developed severe renal failure and died within 48 hours of reperfusion. In contrast, low-TF, hirudin-treated C57BL/6, and PAR-1-/- mice were protected from renal failure and had reduced mortality, tubular injury, neutrophil accumulation, and lower levels of the chemokines KC and MIP-2. Importantly, PAR-1-/- mice had lower chemokine levels despite up-regulation of TF and fibrin deposition. In addition, treating PAR-1-/- mice with hirudin conferred no additional benefit. Somewhat surprisingly, PAR-2 deficiency did not protect from renal failure. These experiments indicate that increased TF activity after renal IR leads to increased CXC chemokine expression and subsequent neutrophil-mediated injury predominantly by thrombin-dependent PAR-1 signaling.     

Dendritic cell depletion exacerbates acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure in the United States and is primarily mediated by toxic metabolites that accumulate in the liver upon depletion of glutathione stores. However, cells of the innate immune system, including natural killer (NK) cells, neutrophils, and Kupffer cells, have also been implicated in the centrilobular liver necrosis associated with APAP. We have recently shown that dendritic cells (DCs) regulate intrahepatic inflammation in chronic liver disease and, therefore, postulated that DC may also modulate the hepatotoxic effects of APAP. We found that DC immune-phenotype was markedly altered after APAP challenge. In particular, liver DC expressed higher MHC II, costimulatory molecules, and Toll-like receptors, and produced higher interleukin (IL)-6, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α). Conversely, spleen DC were unaltered. However, APAP-induced centrilobular necrosis, and its associated mortality, was markedly exacerbated upon DC depletion. Conversely, endogenous DC expansion using FMS-like tyrosine kinase 3 ligand (Flt3L) protected mice from APAP injury. Our mechanistic studies showed that APAP liver DC had the particular capacity to prevent NK cell activation and induced neutrophil apoptosis. Nevertheless, the exacerbated hepatic injury in DC-depleted mice challenged with APAP was independent of NK cells and neutrophils or numerous immune modulatory cytokines and chemokines. Conclusion: Taken together, these data indicate that liver DC protect against APAP toxicity, whereas their depletion is associated with exacerbated hepatotoxicity.     

TLR介导的Nrf2抗氧化通路在对乙酰氨基酚药物性肝损伤中的保护作用

目的研究内毒素(LPS)及Toll样受体(TLR)在对乙酰氨基酚(APAP)药物性肝损伤中的保护作用及其相关机制。方法雄性C57BL/6小鼠40只,分为4组,每组10只。空白对照组腹腔注射0.9%氯化钠溶液,LPS组腹腔注射LPS 10μg/kg,APAP组腹腔注射APAP 300 mg/kg,LPS+APAP组在APAP造模前16 h给予LPS 10μg/kg预处理。通过比较各组血清ALT和AST水平,并通过HE染色评价肝组织损伤程度,观察LPS对小鼠肝损伤的保护作用。测定相应时间点的肝脏组织丙二醛(MDA)、还原型谷胱甘肽(GSH)的变化以及肝组织DHE染色,评价小鼠氧化应激水平。应用Western印迹及RT-PCR检测肝脏Nrf2,Gclc及HO-1的表达水平。结果 LPS预处理可明显减轻APAP所致的肝脏氧化应激反应及肝损伤程度。LPS预处理组的小鼠血清ALT(518.3±142.3对4542±498.4 U/L)、AST(643.3±105.6对5432.1±569.2 U/L)水平及肝组织MDA(78.0±14.5对141.7±26.4 mmoL/mg)水平与模型组相比明显降低,而GSH(6.2±1.7对3.5±1.0μmol/g)水平明显升高(P0.05),肝组织病理损伤明显减轻。同时,LPS预处理可明显促进Nrf2及其下游抗氧化基因的表达。结论 LPS在小鼠APAP肝损伤中起到保护作用,作用机制与Nrf2抗氧化通路的激活相关,可能成为药物性肝损伤的新的治疗策略。     

Deoxyribonuclease 1 aggravates acetaminophen-induced liver necrosis in male CD-1 mice

An overdose of acetaminophen (APAP) (N-acetyl-p-aminophenol) leads to hepatocellular necrosis induced by its metabolite N-acetyl-p-benzoquinone-imine, which is generated during the metabolic phase of liver intoxication. It has been reported that DNA damage occurs during the toxic phase; however, the nucleases responsible for this effect are unknown. In this study, we analyzed the participation of the hepatic endonuclease deoxyribonuclease 1 (DNASE1) during APAP-induced hepatotoxicity by employing a Dnase1 knockout (KO) mouse model. Male CD-1 Dnase1 wild-type (WT) (Dnase1+/+) and KO (Dnase1-/-) mice were treated with 2 different doses of APAP. Hepatic histopathology was performed, and biochemical parameters for APAP metabolism and necrosis were investigated, including depletion of glutathione/glutathione-disulfide (GSH+GSSG), beta-nicotinamide adenine dinucleotide (NADH+NAD+), and adenosine triphosphate (ATP); release of aminotransferases and Dnase1; and occurrence of DNA fragmentation. As expected, an APAP overdose in WT mice led to massive hepatocellular necrosis characterized by the release of aminotransferases and depletion of hepatocellular GSH+GSSG, NADH+NAD+, and ATP. These metabolic events were accompanied by extensive DNA degradation. In contrast, Dnase1 KO mice were considerably less affected. In conclusion, whereas the innermost pericentral hepatocytes of both mouse strains underwent necrosis to the same extent independent of DNA damage, the progression of necrosis to more outwardly located cells was dependent on DNA damage and only occurred in WT mice. Dnase1 aggravates APAP-induced liver necrosis.     

Effects on coagulation and fibrinolysis induced by influenza in mice with a reduced capacity to generate activated protein C and a deficiency in plasminogen activator inhibitor type 1

Influenza infections increase the risk of diseases associated with a prothrombotic state, such as venous thrombosis and atherothrombotic diseases. However, it is unclear whether influenza leads to a prothrombotic state in vivo. To determine whether influenza activates coagulation, we measured coagulation and fibrinolysis in influenza-infected C57BL/6 mice. We found that influenza increased thrombin generation, fibrin deposition, and fibrinolysis. In addition, we used various anti- and prothrombotic models to study pathways involved in the influenza-induced prothrombotic state. A reduced capacity to generate activated protein C in TM(pro/pro) mice increased thrombin generation and fibrinolysis, whereas treatment with heparin decreased thrombin generation in influenza-infected C57Bl/6 mice. Thrombin generation was not changed in hyperfibrinolytic mice, deficient in plasminogen activator inhibitor type-1 (PAI-1(-/-)); however, increased fibrin degradation was seen. Treatment with tranexamic acid reduced fibrinolysis, but thrombin generation was unchanged. We conclude that influenza infection generates thrombin, increased by reduced levels of protein C and decreased by heparin. The fibrinolytic system appears not to be important for thrombin generation. These findings suggest that influenza leads to a prothrombotic state by coagulation activation. Heparin treatment reduces the influenza induced prothrombotic state.     

Ethanol binging exacerbates sinusoidal endothelial and parenchymal injury elicited by acetaminophen

BACKGROUND/AIMS: The pathophysiology of binge drinking of ethanol and its potentiation of acetaminophen (APAP) toxicity has received very little attention. To evaluate if ethanol binging sensitizes hepatic sinusoidal endothelial cells (SEC) and liver to APAP toxicity. METHODS: The histopathological responses to APAP were evaluated in the livers of mice gavaged with APAP alone, following a single, week-end type ethanol binge (4 g/kg every 12 h x 5 doses) or three weekly binges. RESULTS: Six hours after APAP, 600 mg/kg elicited severe centrilobular necrosis together with hemorrhagic congestion and infiltration of erythrocytes into the Space of Disse through large gaps that had formed in SEC. There was no evidence of parenchymal injury at 2 h, but gaps already were formed through the cytoplasm of the SEC by coalescence of fenestrae. A single binge followed by 300 mg/kg APAP elicited SEC and parenchymal injury equivalent to 600 mg/kg APAP alone at 2 and 6 h. The responses were exacerbated following three binges. Lower glutathione levels in the liver were shown in ethanol-binged animals. CONCLUSIONS: Ethanol binging increases APAP hepatotoxicity. SEC are an early target for APAP-induced injury and ethanol binging enhances the SEC injury prior to evidence of parenchymal cell injury.     

The role of hypercoagulability in liver fibrogenesis

The development of hepatic fibrosis on a background of chronic liver injury represents a complex disease trait modulated through the interaction of host genetic factors and environmental influences. Early observations that hepatic inflammation and cirrhosis are associated with the presence of microthrombi within the hepatic vasculature and fibrin/fibrinogen deposition were followed by epidemiological studies showing that carriage of the Factor V Leiden (FvL) mutation, protein C deficiency and increased expression of factor VIII are associated with accelerated progression to cirrhosis in a chronic hepatitis C infection. Additional data suggest that these factors may influence fibrogenesis in many forms of chronic liver disease and extra-hepatic fibrotic processes. Drawing evidence both from liver research and studies of fibrogenesis in other organ systems, two hypotheses may explain how activity of the coagulation cascade influences the rate of hepatic fibrogenesis: tissue ischaemia and parenchymal extinction and direct thrombin mediated stellate cell activation via PAR-1 cleavage. Drawing on preclinical and clinical studies we discuss the evidence for a role for coagulation cascade activity in hepatic fibrogenesis and explore the proposed pathogenic mechanisms that lead to stellate cell activation. The corollary of an association between hypercoagulation and increased fibrosis is that interference with the coagulation cascade may reduce hepatic fibrosis. We conclude this article by examining the implications for future therapeutic intervention.     

Protection against acetaminophen-induced liver injury and lethality by interleukin 10: role of inducible nitric oxide synthase

Mechanistic study of idiosyncratic drug-induced hepatitis (DIH) continues to be a challenging problem because of the lack of animal models. The inability to produce this type of hepatotoxicity in animals, and its relative rarity in humans, may be linked to the production of anti-inflammatory factors that prevent drug-protein adducts from causing liver injury by immune and nonimmune mechanisms. We tested this hypothesis by using a model of acetaminophen (APAP)-induced liver injury in mice. After APAP treatment, a significant increase was observed in serum levels of interleukin (IL)-4, IL-10, and IL-13, cytokines that regulate inflammatory mediator production and cell-mediated autoimmunity. When IL-10 knockout (KO) mice were treated with APAP, most of these mice died within 24 to 48 hours from liver injury. This increased susceptibility to APAP-induced liver injury appeared to correlate with an elevated expression of liver proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, and IL-1, as well as inducible nitric oxide synthase (iNOS). In this regard, mice lacking both IL-10 and iNOS genes were protected from APAP-induced liver injury and lethality when compared with IL-10 KO mice. All strains, including wild-type animals, generated similar amounts of liver APAP-protein adducts, indicating that the increased susceptibility of IL-10 KO mice to APAP hepatotoxicity was not caused by an enhanced formation of APAP-protein adducts. In conclusion, these findings suggest that an important feature of the normal response to drug-induced liver injury may be the increased expression of anti-inflammatory factors such as IL-10. Certain polymorphisms of these factors may have a role in determining the susceptibility of individuals to idiosyncratic DIH.     

The role of the tissue factor-thrombin pathway in cardiac ischemia-reperfusion injury

This article focuses on the role of the tissue factor (TF)-thrombin pathway in cardiac ischemia-reperfusion (I/R) injury. We and others have used rabbit models of cardiac I/R injury to show that anti-TF therapy prevents the transient decrease in regional myocardial blood flow, reduces platelet and fibrin(ogen) accumulation, and reduces infarct size. At present, the mechanism by which TF-initiated coagulation contributes to myocardial injury is not established. Inhibition of TF may decrease intravascular fibrin deposition and thrombosis. However, immunohistochemical studies demonstrated that fibrin deposition was predominantly within the myocardium and depletion of fibrinogen did not reduce infarct size. In contrast, inhibition of thrombin reduced infarct size to a similar extent as anti-TF therapy. We propose that the TF-thrombin pathway may contribute to myocardial injury by an additional mechanism that is not dependent on fibrin deposition but involves activation of protease activated receptor 1 (PAR-1) on vascular endothelial cells and cardiac myocytes. Anti-TF therapy would inhibit both thrombin-dependent fibrin deposition and thrombin-dependent PAR-1 signaling.     

Intravascular coagulation in the development of massive hepatic necrosis induced by Corynebacterium parvum and endotoxin in rats

When Escherichia coli endotoxin was intravenously injected into rats given killed Corynebacterium parvum 6 days previously, fibrin deposition and endothelial cell injury occurred in hepatic sinusoids at 1.5 h and were intensified thereafter. Serum alanine aminotransferase values were increased along with prothrombin time and decreased plasma levels of antithrombin III and coagulation factor VIII:C at 5 h. Antithrombin III concentrate (plus heparin) or superoxide dismutase infused concurrently with injection of endotoxin significantly attenuated the derangements of these variables and the histologic extent of liver injury at 5 h. Intravascular coagulation, probably developing through the action of superoxide anion, may contribute to the development of massive hepatic necrosis induced by C. parvum and endotoxin in rats.